Mastitis, an infection of the mammary gland usually caused by bacteria or fungus, results in major economic losses to the dairy industry yearly. Among the bacterial species most commonly associated with mastitis are various species of the genus Streptococcus, including S. aureus, S. uberis, (untypeable), S. agalactiae (Lancefield group B), S. dysgalactiae (Lancefield group C), S. zooepidemicus, and the Lancefield groups D, G., L and N streptococci. Some of those species are contagions (e.g. S. agalactiae), while others are considered environmental pathogens (e.g. S. dysgalactiae and S. uberis). The environmental pathogen S. uberis is responsible for about 20% of all clinical cases of mastitis (Bramley, A. J. and Dodd, F. H. (1984) J. Dairy Res. 51:481-512; Bramley, A. J. (1987) Animal Health Nutrition 42:12-16; Watts, J. L. (1988) J. Dairy Sci. 71:1616-1624); it is the predominant organism isolated from mammary glands during the non-lactating period (Bramley, A. J. (1984) Br. Vet. J. 140:328-335; Bramley and Dodd (1984) J. Dairy Res. 51:481-512; Oliver, S. P. (1988) Am. J. Vet. Res. 49:1789-1793).
Mastitis resulting from infection with S. uberis is commonly subclinical, characterized by apparently normal milk with an increase in somatic cell counts due to the influx of leukocytes. The chemical composition of milk is changed due to suppression of secretion with the transfer of sodium chloride and bicarbonate from blood to milk, causing a shift of pH to a more alkaline level. S. uberis mastitis may also take the form of an acute clinical condition, with obvious signs of disease such as clots or discoloration of the milk and swelling or hardness of the mammary gland. Some cases of the clinical disease can be severe and pyrexia may be present. For a review of the clinical manifestations of S. uberis mastitis, see, Bramley (1991) Mastitis: physiology or pathology. p. 3-9. In C. Burvenich, G. Vandeputte-van Messom, and A. W. Hill (ed.), New insights into the pathogenesis of mastitis. Rijksuniversiteit Gent, Belgium; and Schalm et al. (1971) The mastitis complex-A brief summary. p. 1-3. In Bovine Mastitis. Lea & Febiger, Philadelphia
Conventional antibacterial control methods such as teat dipping and antibiotic therapy are effective in the control of many types of contagious mastitis, but the environmental organisms typically found in all dairy barns are often resistant to such measures. Vaccination is therefore an attractive strategy to prevent infections of the mammary glands, and has been shown to be beneficial in the case of some contagious mastitis pathogens.
The literature is limited regarding vaccination studies with S. dysgalactiae and S. uberis, and variable results have been observed. In some cases, immunization has resulted in increased sensitivity to the specific organism and in other cases strain-specific protection has been obtained.
For example, previous studies have shown that primary infection with S. uberis can considerably reduce the rate of infection following a second challenge with the same strain (Hill, A. W. (1988) Res. Vet. Sci. 44:386-387). Local vaccination with killed S. uberis protects the bovine mammary gland against intramammary challenge with the homologous strain (Finch et al. (1994) Infect. Immun. 62:3599-3603). Similarly, subcutaneous vaccination with live S. uberis has been shown to cause a dramatic modification of the pathogenesis of mastitis with the same strain (Hill et al. (1994) FEMS Immunol. Med. Microbiol. 8:109-118). Animals vaccinated in this way shed fewer bacteria in their milk and many quarters remain free of infection.
Nonetheless, vaccination with live or attenuated bacteria can pose risks to the recipient. Further, it is clear that conventional killed vaccines are in general largely ineffective against S. uberis and S. agalactiae, either due to lack of protective antigens on in vitro-grown cells or masking of these antigens by molecular mimicry.
The current lack of existing mastitis vaccines against S. agalactiae or the contagious streptococcus strains is due at least in part to a lack of knowledge regarding the virulence determinants and protective antigens produced by those organisms which are involved in invasion and protection of the mammary gland (Collins et al. (1988) J. Dairy Res. 55:25-32; Leigh et al. (1990) Res. Vet. Sci. 49: 85-87; Marshall et al. (1986) J. Dairy Res. 53: 507-514).
S. dysgalactiae is known to bind several extracellular and plasma-derived proteins such as fibronectin, fibrinogen, collagen, alpha-II-macroglobulin, IgG, albumin and other compounds. The organism also produces hyaluronidase and fibrinolysin and is capable of adhering to and invading bovine mammary epithelial cells. However, the exact roles of the bacterial components responsible for these phenotypes in pathogenesis is not known.
Similarly, the pathogenesis of S. uberis infection is poorly understood. Furthermore, the influence of S. uberis virulence factors on host defense mechanisms and mammary gland physiology is not well defined. Known virulence factors associated with S. uberis include a hyaluronic acid capsule (Hill, A. W. (1988) Res. Vet. Sci. 45:400-404), hyaluronidase (Schaufuss et al. (1989) Zentralbl. Bakteriol. Ser. A 271:46-53), R-like protein (Groschup, M. H. and Timoney, J. F. (1993) Res. Vet. Sci. 54:124-126), and a cohemolysin, the CAMP factor, also known as UBERIS factor (Skalka, B. and Smola, J. (1981) Zentralbl. Bakteriol. Ser. A 249:190-194), R-like protein, plasminogen activator and CAMP factor. However, very little is known of their roles in pathogenicity.
The use of virulence determinants from Streptococcus as immunogenic agents has been proposed. For example, the CAMP factor of S. uberis has been shown to protect vertebrate subjects from infection by that organism (Jiang, U.S. Pat. No. 5,863,543).
They antigen of the group B Streptococci strain A909 (ATCC No. 27591) is a component of the c protein marker complex, which additionally comprises an α and β subunit (Boyle, U.S. Pat. No. 5,721,339). Subsets of serotype Ia, II, and virtually all serotype Ib cells of group B streptococci, have been reported to express components of the c protein. Use of the γ subunit as an immunogenic agent against infections by Lancefield Group B Streptococcus infection has been proposed. However, its use to prevent or treat bacterial infections in animals, including mastitis in cattle, has not been studied.
A GapC plasmin binding protein from a strain of Group A Streptococcus has previously been identified and characterized, and its use in thrombolytic therapies has been described (Boyle, et al., U.S. Pat. No. 5,237,050; Boyle, et al., U.S. Pat. No. 5,328,996). However, the use of GapC as an immungenic agent to treat or prevent mastitis was neither described nor suggested.
The group A streptococcal M protein is considered to be one of the major virulence factors of this organism by virtue of its ability to impede attack by human phagocytes (Lancefield, R. C. (1962) J. Immunol. 89:307-313). The bacteria persist in the infected tissue until antibodies are produced against the M molecule. Type-specific antibodies to the M protein are able to reverse the antiphagocytic effect of the molecule and allow efficient clearance of the invading organism.
M proteins are one of the key virulence factors of Streptococcus pyogenes, due to their involvement in mediating resistance to phagocytosis (Kehoe, M. A. (1991) Vaccine 9:797-806) and their ability to induce potentially harmful host immune responses via their superantigenicity and their capacity to induce host-cross-reactive antibody responses (Bisno, A. L. (1991) New Engl. J. Med. 325:783-793; Froude et al. (1989) Curr. Top. Microbiol Immunol. 145:5-26; Stollerman, G. H. (1991) Clin. Immunol. Immunopathol. 61:131-142).
However, obstacles exist to using intact M proteins as vaccines. The protein's opsonic epitopes are extremely type-specific, resulting in narrow, type-specific protection. Further, some M proteins appear to contain epitopes that cross react with tissues of the immunized subject, causing a harmful autoimmune response (See e.g., Dale, J. L. and Beached, G. H. (1982) J. Exp. Med 156:1165-1176; Dale, J. L. and Beached, G. H. (1985) J. Exp. Med. 161:113-122; Baird, R. W., Bronze, M. S., Drabs, W., Hill, H. R., Veasey, L. G. and Dale, J. L. (1991) J. Immun. 146:3132-3137; Bronze, M. S. and Dale, J. L. (1993) J. Immun 151:2820-2828; Cunningham, M. W. and Russell, S. M. (1983) Infect. Immun. 42:531-538).
An octavalent M protein vaccine has been constructed and was tested for protective immunogenicity against multiple serotypes of group A streptococci infection in rabbits. However, the immune response obtained was serotype-specific, conferring protection only against those bacterial strains exhibiting the M protein epitopes present in the chimeric protein (Dale, J. B., Simmons, M., Chiang, E. C., and Chiang, E. Y. (1996) Vaccine 14:944-948).
Chimeric proteins containing three different fibronectin binding domains (FNBDs) derived from fibronectin binding proteins of S. dysgalactiae and Staphylococcus aureus have been expressed on the surface of Staph. carnosus cells. In the case of one of these proteins, intranasal immunizations with live recombinant Staph. carnosus cells expressing the chimeric protein on their surface resulted in an improved antibody response to a model immunogen present within the chimeric surface protein.
A chimeric Protein G molecule (a type III Fc binding protein specific for the Fc region of all subclasses of IgG antibody molecules) is known, but its use as an immunogenic agent has not been described or suggested (Bjorck, et al. (1992) U.S. Pat. No. 5,108,894).
Until now, the protective capability of GapC multiple epitope fusion proteins has not been studied.